Chemistry: molecular biology and microbiology – Virus or bacteriophage – except for viral vector or...
Patent
1997-08-22
2000-02-22
Hutzell, Paula K.
Chemistry: molecular biology and microbiology
Virus or bacteriophage, except for viral vector or...
435 5, 435 6, 435 71, 435 694, 536 234, C12N 700, C12G 170, C12G 168, G01N 3353
Patent
active
060279303
DESCRIPTION:
BRIEF SUMMARY
TECHNICAL AREA OF INVENTION
This invention relates to the selection of specific binding proteins and their genes using a new mutant bacteriophage in phage display techniques.
BACKGROUND OF THE INVENTION
In our patent application PCT/SE94/01166 we described a method of selection of antigenic or other specific binding protein molecules, and their encoding DNA, by means of a phage display package produced by co-expression of a phagemid vector and a helper phage, in which the DNA encoding the binding peptide is inserted into the phagemid vector so as to be expressed as a fusion with a truncated phage gene III protein. The resulting display package lacks functional protein III, and is therefore not infectious, but it can be selectively rendered infectious by means of a fusion protein comprising functional protein III and the complementary binding partner for the specific binding protein present on the surface of the phage display package. The function of the helper phage is to provide the genes necessary for packaging the expression products of the phagemid; but it will be deficient in gene III so that the resulting phage display package will not contain functional gene III.
A similar system has been disclosed also in example 2 of EP-A-614989. In that case, the helper phage used was a derivative of phage Fd tet, known as fKN16, which had a deletion of 507 nucleotides in gene III. In our PCT application we used a derivative of phage M13 KO7 having a 1121 nucleotide deletion in gene III between nucleotides 1525 and 2646.
We have found that this M13 helper phage is considerably better than phage fKN16 in the performance of the procedure. Not only that, we have now also found that it is similarly better than another Fd phage (fCA55) having a larger deletion in gene III; and furthermore, that we can improve on our M13 phage construction previously described.
The experimental basis for this is presented below. In summary, however, the following general features seem to emerge.
DEFINITION OF THE INVENTION
Firstly, whereas in our original delta 3 version of the M13 helper phage, we had deleted the gene III promoter and most of the coding sequence, in our new delta 3.2 version we kept the gene III promoter but deleted effectively all the gene III coding sequence. Both constructs had the effect of preventing expression of any gene III protein, but the delta 3.2 version, by keeping the gene III promoter, retained the small intergenic (IG) region between gene VIII ligand gene III. It appears that this is at least in part responsible for the improved performance of the delta 3.2 helper phage over the delta 3 phage. Accordingly, the design of a helper phage for use in this type of selection procedure should take account of this, and substantially retain the small IG region between gene VIII and gene III.
It may not be necessary to retain the entire small IG region. This region contains a number of functional motifs. For example, as well as the promoter for gene III, there is at least one transcription termination sequence. Precisely what aspect of the small IG region is important remains to be seen, but the minimum sequence required from this region could if desired be determined by routine experimentation.
Secondly, while this development has been discovered in connection with the M13 phage, it seems likely that a similar phenomenon will be observed with other filamentous helper phages. Examples of such are fd, f1, 1f1, 1ke, ZJ/2, Xf; Ff, Pf1 and Pf3. Thus, optimal function of for example Fd-type phages in this procedure might require retention of the small IG region (along with other features).
Thirdly, insofar as retention of the small IG region involves retention also of the promoter for gene III, it is probably important that there be no gene III coding sequence, or even perhaps other coding sequence, expressible from the transcript from that promoter. This would suggest deletion of all gene III coding sequence and/or ensuring that there is no translational start codon in the transcript such as would give a prote
REFERENCES:
Duenas et al. Bio/Technology. 12(10) 999-1002, Oct. 1994.
FEMS Microbiology Letters, vol. 125, 1995, Marta Duenas et al, "Novel helper phage design: intergenic region affects the assembly of bacteriophages and the size of antibody libraries" p. 317-p.322.
Crissman and Smith, "Gene-III Protein of Filamentous Phages: Evidence for a Carboxy-Terminal Domain with a Role in Morphogenesis", Virology 132: 445-455 (1984).
Barbas III et al, "Assembly of combinatorial antibody libraries on phage surfaces: The gene III site", Proc. Natl. Acad. Sci USA 88:7978-7982 (1991).
Markland et al, "Design, construction and fuction of a multicopy display vector using fusions to the major coat protein of bacteriophage M13", Gene 109:13-19 (1991).
BioInvent International AB
Hutzell Paula K.
Sun-Hoffman Lin
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